2-Photon Imaging

Laser Safety

From the Spectra-Physics manual: The Spectra-Physics Tsunami and its pump laser, the Millenia V_s are Class IV-High Power Lasers, whose beams are, by definition, safety and fire hazards.

Take precautions to prevent exposure to both direct and reflected beams. Diffuse as well as specular reflections cause severe eye or skin damage.

Because the Tsunami laser emits cw (continuous wave) and pulsed infrared radiation, it is extremely dangerous to the eye. Infrared radiation passes easily through the cornea, which focuses it on the retina, where it can cause instantaneous permanent damage.

Precautions:

Wear protective eyewear at all times.

Maintain a high ambient light level in the laser operation area. This keeps the eye's pupil constricted, thus reducing the possibility of eye damage.

Avoid looking at the output beam; even diffuse reflections are hazardous.

Avoid blocking the output beam or its reflections with any part of the body.

Use an infrared detector to verify that the laser beam is off before working in front of the laser.

Avoid wearing jewelry or other objects that may reflect or scatter the beam while using the laser.

Keep the cover(s) on all equipment.

Setting up the software:

Unless you are trained to start the 2-photon system, you must arrange with me to have the Ti:sapphire laser ready to go. Use the same startup instructions as for the confocal - turning on only the lasers needed.

Select "2P" from the filter list.

IR laser is active.

Start with a neutral density of ND6.

Substrat should be selected as the dichroic for 2-photon excitation only. If combining the 2-photon with confocal excitation then select the same dichroic used for the confocal. Do not change dichroics during acquisition.

Check the same features as with confocal scanning:

Adjust the slits for the emission spectrum of the fluorophore(s) excited with the two photon laser.

Select the Stain (if available) to indicate the emission spectrum.

Select the Clut, or color table, for scanning.

Activate the PMT(s) - PMT1 may be bad - we are waiting for a replacement (9/21). Use PMT 2 for DAPI.

The objective used for scanning needs to be selected in the software.

The pinhole should be open all the way, it is not used for 2-photon imaging. "Open all the way" will depend on the selected objective lens.

Continue setting up the system for only 2-photon acquisition.

Go to the section on collecting sequential scans if combining 2-photon and confocal acquisition.

Setting up the software:

Select "2ph" from the filter list.

IR laser is active.

Start with a neutral density of ND6.

Check the same features as with confocal scanning:

Adjust the slits for the emission spectrum of the fluorophore(s) excited with the two photon laser.

Select the Stain (if available) to indicate the emission spectrum.

Select the Clut, or color table, for scanning.

Activate the PMT(s) - PMT1 may be bad - we are waiting for a replacement (9/21). Use PMT 2 for DAPI.

Check that the software is correctly reading the objective.

The pinhole should be open all the way, it is not used for 2-photon imaging. "Open all the way" will depend on the selected objective lens.

If you are combining 2-photon with confocal acquisition, then it is often easier to collect the images sequentially. Go to the section on collecting sequential scans.

Simultaneous collection of 2-photon and confocal images may work but check for crosstalk in each channel. A pinhole is usually not used for 2-photon imaging so the image may be too dim when collected at the same pinhole settings used for the confocal. Be careful with the neutral density settings - it is easy to overheat the sample with too much power.

Continue setting up the system for only 2-photon acquisition.